Structure of the AlgKX modification and secretion complex required for alginate production and biofilm attachment in Pseudomonas aeruginosa

Synthase-dependent secretion systems are a conserved mechanism for producing exopolysaccharides in Gram-negative bacteria. Although widely studied, it is not well understood how these systems are organized to coordinate polymer biosynthesis, modification, and export across both membranes and the peptidoglycan. To investigate how synthase-dependent secretion systems produce polymer at a molecular level, we determined the crystal structure of the AlgK-AlgX (AlgKX) complex involved in Pseudomonas aeruginosa alginate exopolysaccharide acetylation and export. We demonstrate that AlgKX directly binds alginate oligosaccharides and that formation of the complex is vital for polymer production and biofilm attachment. Finally, we propose a structural model for the AlgEKX outer membrane modification and secretion complex. Together, our study provides insight into how alginate biosynthesis proteins coordinate production of a key exopolysaccharide involved in establishing persistent Pseudomonas lung infections.

The work by Gheroghita et al. describes the structure of the AlgKX complex from Pseudomonas putida that reveals important insight into the modification and export apparatus of synthasedependent secretion systems. This work required the crystallization of these proteins from a different species (previously only P. aeruginosa and fluorescens had been used) and is a remarkable feat in itself, as it delineates for the first time important residues in the binding surface (which previously had been a mystery) and uncovers an electronegative groove that further supports the biological relevance of this interaction in the export of alginate. Importantly, the manuscript also outlines the functional relevance of this interaction by determining the association constants of the proteins for each other and for alginate-specific (M and MG) polymers. In a feat of biological strength, these researchers then systematically underscored these results using enzymatic (acetylesterase), protein-protein interaction (co-immunoprecipitation) and phenotypic mutant (involving complementation with protein variants) assays. Together, these results substantially advance our understanding of protein complex formation (AlgEKX), the binding/secretion of alginate (polymer type, the importance of the N-terminus of AlgX and binding surface extension across AlgEKX) and the order of modification (epimerization prior to acetylation) prior to export.

Revision suggestions:
Main Comments: 1. For the AlgX N-terminal interactions (Lines123-128), the AlgX side chains of E35 and C37 do not appear to be conserved based on your coloring scheme. For AlgK, the conservation (both main and side chain) seems more clear (aside from 404). Since these side chains are not conserved, what does this mean for the relevance of the side-chain interactions in this model? Despite not being conserved are there similar possible residues that could form a link at this position in other Pseudomonas species sequences? As it stands, the lack of conservation calls into question the relevance of these side chains and may need some rewording to account for this……but I should mention that the other conserved residues outweigh the discrepancy with these few residues. On L127, the use of the term "variably conserved" might also need changing since it could be a bit misleading given the side chain conservation issue. L127-128 -"the side chain of S234 and main chain of Y235 interact with A" -Again, the relevance of this needs to be underscored by the conservation coloring scheme that you have used. It seems that Y235 main chain is the most important of these given the conservation?
2. Poly MG Association Constants (L183-184) -I agree that these numbers are roughly double that of Poly M for 2 of the 3 (ie. not GMGGM). This is above the standard error and appears to be significant. However, you might want to be careful in over-interpreting this difference of in vitro numbers that are less than an order of magnitude different. It should also be kept in mind that the polymer lengths are different and it would have been more ideal to test the same lengths. I understand that these polymers can be problematic to make/isolate and it might not always be possible to do this experiment ideally, but perhaps some wording to account for this variability would be beneficial for future readers. As noted later in the document (Discussion), the results that the acetyltransferase activity was not affected by polyM or polyMG substrates supports the need to not over-interpret the association constant results. Some minor rewording in key places could easily emphasize this and shouldn't be a major issue. Once things show up in the literature it can be hard to reconcile with further contradictory data, so being mindful of this with the wording should help to avoid issues in the future.
Other Specific Minor Comments: L51-biofilm attachment -this might be personal preference, but biofilm formation seems more appropriate L79-81 -a mention here about the known/unknown 1) levels of acetylation and epimerization; and 2) order to the modifications would be beneficial to keep the current context for the reader. This also will help to place the depiction of Fig. 1a in the proper context. L87-88 -I agree that there is a gap. This introduction is fairly brief and to the point (which I like), but I think that it could be expanded here with some details as to what is known about AlgK and AlgX in the complex a bit more. A couple lines (or a short paragraph) summarizing the relevant literature on these proteins would be sufficient to lay the foundation for past work that has left the gap that this manuscript will fill. L99-100 -suggest removing the closing sentence, as it is a bit redundant by the end of the paragraph L112-113 -model 9 TPRs (R3-10) -this seems like 8 TPRs and looks like it in Figure 1c L119 -"its" should be "it's" and there is a missing "that" after the (residues 30-37) L120 -How was the interface calculated? I assume it was with PISA based on the methods, but could you add the program in here to for clarity for the reader. L184 -remove GMGGM from the text as the two association constant values are here are for the other MG polymers and not for this one. L195 -This is a great graph, but out of interest sake….when the rates of K and X alone are added together, does that equal KX? What about for the separate trails of polymer+K+X, does that equal KX in the presence of polymer. Is this really an increase or just an additive amount? L207 -suggest replacing "Thus" with "Nonetheless" for better flow L229 -This is more because I am familiar with these plasmids and was interested in the expression system, but how is AlgX making it to the periplasm if the N-terminus is missing? It looks like the expression background (as per the methods section L659) is pET24b and not pET26b (which would have the pelB signal sequence). Perhaps a bit more clarity here would help. L285 -"our model suggests that polymer acetylation immediately precedes export" -At least for AlgX, but it seems that there is still uncertainty as to how AlgIJF fit into the picture. Could this be earlier or at a different site in the export process? The way this statement reads is that AlgIJF are not part of the picture. L285-288 -This is only a comment, but this sentence sums up very strong evidence in support of the model and provides great insight into this process. Definitely a worthwhile finding. L300 -"preferentially binds polyMG ligands" -As noted above…..Some, but not GG ligands? L319 -I am still not sure that this is a large difference, although polymer binding of itself is significant, the fact that the rate of acetylation wasn't affected suggests that both polyM and MG are within the same binding range of each other to be biologically equal. It is the order within the complex that likely determines that AlgG comes before AlgX activity. Especially given the new information that AlgK does not bind AlgG. L346 -This is only a suggestion, but starting a new paragraph at "At the inner membrane" might be helpful with the flow for the reader. L624 -reword "flanking the outside, flanking regions" Figure 1 -"hydrogen bonds and salt bridge interactions represented by yellow and pink lines" -The pink lines are not evident here. Perhaps adjusting the text or the image would help. Figure 2 -In panel D, Can the sizing of the gels be made to more closely match the panels in B for easier comparison sake. Perhaps this was shifted in the PDF version. Also, this is a suggestion, but the use of color in the figure is not entirely necessary. For the chromatograms, the use of solid and dashed lines would be as effective and may be clearer to colorblind readers. Figure 3C -Bars should extend under the PolyM alone and PolyMG alone at the bottom of the graph to denote the series better. Figure 4a -This is a bit unclear since AlgL and AlgX are similar in size and you can't see both bands on the blots above. Perhaps this just needs some clarification.
Extended Data Table 1 -A couple of things: 1. Tables should have a clear title. The "values in parentheses" is better in the legend below the table. 2.Referencing the formulas used for RMerge and RWork calculations would be nice 3. I assume that you have listed the number of unique reflections here, can you verify this and make it clearer? Also, how many were in the high resolution shell? 4. The Ramachandran statistics (favored and allowed) are mentioned in the text, but often they are included here too. I know this is an extended table but including the standardized parameters that are commonly listed in Table 1 would be beneficial.
Supplementary Fig. 3&4 -This likely only bothers the chemists among us, but the bond lengths between the C1 and C4 linkages are not consistent. For example, in figure 3 between every third and fourth sugar (starting from the left) the linkage is off and leads to a structure that at first glance is a bit skewed. I suspect that this figure was made by creating a small multimer that was then copied and joined. No problem with that as long as the bond lengths/angles are consistent. Can this be fixed? Also, the C2-OH group runs into the ring oxygen and is hard to read.
Reviewer #3 (Remarks to the Author): The alginate exopolysaccharide plays a crucial role in the infection by P. aeruginosa. In this manuscript, Gheorghita et al. reported the crystal structure of the AlgKX complex from Pseudomonas putida involved in alginate exopolysaccharide acetylation and export; they found The N-terminus of AlgX is required for complex formation with AlgK, and that formation of the complex is vital for polymer production and biofilm attachment; they further demonstrate that AlgKX preferentially binds modified alginate oligosaccharides. Overall, the authors did a solid job in structure determination of the AlgKX complex and functional analysis of the AlgK-AlgX interactions in alginate production and substrate preference. However, the reviewer thinks the following points need to be addressed by the authors.
1. The authors reported the structure of AlgKX from P. putida, yet functional analysis was conducted in P. aeruginosa, sequence alignments of the two proteins from different species should be included, at least, in supplementary data. 2. Line 196, how to explain that AlgKX prefers to bind polyMG yet not increase the acetylesterase activity? Does polyMG promote the formation of AlgKX complex? 3. To obtain the crystal of the AlgKX complex, the authors mixed the purified AlgK and AlgX at a 1:1 molar ratio before crystallization. Just curious, no further size exclusion chromatography is needed? Co-expression and co-purification of the AlgKX complex are not successful? 4. The AlgKX complex is mainly formed by the interaction between the N-terminus of AlgX (residues 30-37) and the TPRs R9-R10 of AlgK. To investigate the important role of the N-terminus residues of AlgX, some point mutations of this N-terminus could be tested to pinpoint the most critical residues in this region for the interaction. 5. Fig. 2a, there are two peaks in the gel filtration chromatograms of AlgKX, and the SDS gel shows that AlgX and AlgK are both in the two peaks. It makes more sense if peak 2 is the excessively one sample of either AlgX or AlgK. &$ )86$ %3" @74 :86>0@8<; <5 '96/F,@4>: 0;3 '96+ <; -(-#649 0>4 ?0:4$ *@ 8? 14@@4> @< 8;29A34 .4?@4>; 19<@? @< ?7<C '96/F,@4>: 0;3 '96+" >4?=42@8B49E$ 7. Line 250, to conclude that the formation of the AlgKX complex is required for alginate =><3A2@8<;" @74 0A@7<>? ;443 @< 4D29A34 @74 =<??81898@E @70@ @74 '96/F,@4>: 9<?8;6 8@? acetylesterase activity affects the alginate production. 8. Fig. 3b, the peaks and labels of AlgKX-ligand in ESI mass spectra are confused. The red label is "AlgKX", but the black numbers may indicate the peaks of AlgKX and the red circles indicate the peaks of AlgKX-ligand. 9. The authors used AlphaFold2 to generate a model of the AlgEK. Does Alphafold2 predict the AlgKX structure with high confidence? 10. In the proposed model of the AlgEKX modification and secretion complex, where is the energy for alginate secretion from? The authors should speculate this, at least in discussion. 11. Extended Data Table 1, numbers in parentheses are not stated.